Benzene, an environmental pollutant, is myelotoxic and leukemogenic in humans. The molecular mechanisms that can account for its biological effects have not been fully elucidated. We hypothesize that one of the underlying mechanism involves nitration of proteins by peroxynitrite and/or by bone marrow myeloperoxidase-dependent pathways in nitric oxide (NO) metabolism. Using 3-nitrotyrosine [Tyr(NO(2))] as a biomarker for NO-induced damage to proteins, we examined the effects of benzene on the levels of Tyr(NO(2)) in bone marrow in vivo. Groups of 8 weeks old B6C3F(1) male mice were given a single i.p. injection of benzene (50, 100, 200 or 400mg/kg bodyweight) in corn oil. The mice in control groups received either no treatment or a single injection of the vehicle. The mice were killed 1h after treatment and proteins were isolated from bone marrow, lung, liver and plasma. The proteins were enzymatically hydrolyzed; amino acids were separated and purified by high pressure liquid chromatography, derivatized, and quantified by electron capture-negative chemical ionization-gas chromatography/mass spectrometry (EC-NCI-GC/MS). In the GC/MS assay, 3-nitro-l-[(13)C(9)]tyrosine was used as an internal standard and l-[(2)H(4)]tyrosine served to monitor artifactual formation of 3-nitrotyrosine during sample preparation and analysis. We found that treatment of mice with benzene elevates nitration of tyrosine residues in bone marrow proteins. There was a dose (50-200mg benzene/kg b.w.)-dependent increase in protein-bound Tyr(NO(2)) formation (1.5- to 4.5-fold); however, the levels of Tyr(NO(2)) at 400mg benzene/kg b.w. were significantly higher than control but lower than that formed at 200mg benzene/kg b.w. The results of this study, for the first time, indicate that benzene increases protein-bound 3-Tyr(NO(2)) in bone marrow in vivo, and support our previous finding that benzene is metabolized to nitrated products in bone marrow of mice; collectively, these results may in part account for benzene-induced myelotoxicity.